Methods and preparations of the latex from the croton species

ABSTRACT

The invention herein describes a procedure for organically extracting the lipophilic components from plants of the Family Euphorbaciae, specifically but not limited to the genus  Croton . The extraction (CGO 110), is deplete of the normal proanthocyanidin content found in the parent material, yet retains its pharmacocological abilities and unlike the parent material, is selectively cytotoxic to cancer cells. The depletion of the proanthocyanidin components makes the product more amenable to preparations for the benefit of ameliorating both human and animal disease.

This application claims benefit of U.S. Provisional Application Ser. No.60/416,751 filed on Oct. 5, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Sangre de grado or Sangre de drago, also known as “Dragon's Blood,” is aviscous latex sap derived from the bark of various Croton species (C.dracanoides, C. erythrochilus, C. gossypifilius, C. lechleri, C.palanostigma, C. sakutaris and C. urucurana) indigenous to the SouthAmerican rainforests. This latex has a deep red or burgundy color thatis attributed to its substantial proanthocyanidin content, estimated tobeing approximately 90% of the solid constituents of the sap.Ethnomedically, the latex is topically applied for the treatment of painand itching associated with insect bites and stings, as well as plantreactions. It is applied to the gums of patients after toothextractions, is utilized as a vaginal wash in the case of excessivebleeding and in the treatment of herpes where it is applied topically.It is also applied to open wounds as an anti-infective and as acicatrizant to accelerate the healing process. This latter effect mayresult from its constitutive taspine and crolechleric acid. It is takeninternally for a variety of distressing gastrointestinal symptoms,including the treatment of diarrhea, ulcers, vomiting and gutinflammation, as well as throat infections, tuberculosis and rheumatism.Oral intake is also associated with the ethnomedical application forcancer.

These traditional applications within South America cultures are lesslikely to be used in the Western world because of several constraints.Primarily, Sangre de grado's intense color, as suggested by thereference to blood in its name, limits its ability to be used topically.In addition, Sangre de grado discolors and stains clothing in a similarmanner as red wine, another proanthocyanidin rich extract. A means ofreducing the proanthocyanidin content (and hence color) of the latexwhilst retaining its useful biological properties would represent asignificant improvement over the traditional botanical and allow formore widespread application.

The proanthocyanidins have been implicated as the mediators of Sangre degrado's antidiarrheal properties through the prevention of cAMP mediatedepithelial secretion. However, recent evidence suggests that Sangre degrado attenuates these epithelial secretory mechanisms by preventing theactivation of sensory afferent nerves that promote diarrhea, localinflammation, edema, as well process signals to the brain for pain,nausea and itching. Capsaicin, the active component of chili peppers,stimulates these sensory afferent nerves and Sangre de grado has beenshown to impair capsaicin-induced epithelial secretion of electrolytes.

SUMMARY OF THE INVENTION

Aspects of the invention are summarized below to aid in theunderstanding of embodiment(s) of the invention and the application.Yet, the invention is fully defined by the claims of the application.

The latex or sap derived from the bark of the Croton species of theSouth American rainforests is associated with various ethnomedicalapplications including the treatment of cancer, diarrhea,gastrointestinal distress, pain and itching. While effective for theseindications the traditional ethnomedicine has undesirable effects thatlimit its use.

The present invention generally comprises an extract composition derivedfrom the latex or sap of the Croton species that retains desirablemedicinal benefits despite reduced proanthocyanidin content. The extractdisclosed herein retains the ability to inhibit emesis and activation ofsensory afferent nerves. The extract furthermore discriminately promotescancer cell death, unlike the parent material, at concentrations thatfail to promote cell death in normal cells. The extract compositionretains its desirable properties but has a reduced cytotoxicitysignifying an improvement over the parent botanical.

The extract composition is further incorporated into a biologicallyactive dosage unit forming a beneficial wound-healing composition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The extraction process significantly (“*”) reduces theproanthocyanidin content of the parent latex (SdG) from the speciesCroton lechleri from the family Euphorbaceae. When combined in a basevehicle, such as Aloe barbadensis shown here, the extract (CGO 110)produced a mixture absent of the intense color seen in similarpreparations with the parent latex. This change, which is readilyquantifiable by spectrophotometer, negates the discolorizing (i.e.staining) properties commonly associated with proanthocyanidins and theparent latex and allows for practical dermatological preparations;

FIG. 2. The prototypical activator of sensory afferent nerves,capsaicin, was topically applied to the mucosal surface of the stomachin anesthetized rats and mucosal blood flow measured by a Laser DopplerFlow meter. The marked increase in mucosal blood flow induced by 300 μMcapsaicin was prevented by either the parent material, SdG, or itsorganic extract, CGO 110 deplete of proanthocyanidins at doses of 2 and0.2 mg/ml, respectively, indicating that the organic extract retains theability to effectively prevent the activation of sensory afferentnerves;

FIG. 3. Using a well-established ferret model of post-operativecomplications of nausea, emesis and itch induced by morphine, theorganic extract CGO 110 was administered intraperitoneally (3 mg/kg) toferrets 15 minutes prior to the subcutaneous injection of 0.05 mg/kg ofmorphine-6-glucuronide (M6G). Administration of M6G caused a significantnumber of vomiting (2.2±0.4) and retching (10±1.2) incidences in thecontrol group while in those animals pre-treated with CGO 110, thenumber of these episodes was virtually abolished (vomiting 0.6±0.3;retching 2.2±0.7, P<0.05). It is clear that this organic extractionprocedure contains active components and is effective in the treatmentof emesis and itch.

FIG. 4. The selective cytotoxic ability of CGO 110 was tested in vitroin cancerous cells from the gastrointestinal tract (AGS: stomach) andalso in both normal macrophages and normal intestinal epithelial cells(IEC-18). In normal cells, Sangre de grado caused significant cell deathin both macrophages and IEC-18 cells while the same concentrations ofthe organic extract CGO 110 did not. In stomach cancer cells (AGS), bothCGO 110 and Sangre de grado were cytotoxic and the extract was morepotent than the parent botanical. Treatment of stomach cancer cells(AGS) with both CGO 110 and Sangre de grado caused cytotoxicity (celldeath), and the lipidic extract, CGO 110, was more potent than theparent botanical [the “*” denotes a significant difference between theSangre de grado and organic extract CGO 110 formulations (P<0.05)].Collectively, these results indicate that CGO 110 is selectivelycytotoxic to cancerous cells compared to the parent botanical, therebyrepresenting a marked improvement in safety.

DESCRIPTION OF AN EMBODIMENT

Extraction Procedure

According to one aspect of this invention, a process that retains andconcentrates the lipophilic components while reducing the hydrophilicproanthocyanidin content of the plant material resolves a familyEuphorbaciae extraction. This extraction process significantly reducesthe extracted composition of the hydrophilic proanthocyanidins, andhence its intense burgundy color, making it more amenable to topicalhealth care preparations. Furthermore, a product of this lipidicextraction, is embodied in CGO 110 from the species Croton lechleri fromthe family Euphorbaceae, is selectively cytotoxic to cancerous cells,unlike the parent material, representing an improvement in safety andsuggesting applications in the treatment of cancerous cells. Preferredmethods to accomplish the aforementioned family Euphorbaceae extractionare described by the procedures below but it is contemplated that askilled practitioner could device obvious variations of the proceduresgiven the disclosure herein and the desired results.

Extraction Process 1.

Latex, or sap from Croton species is mixed with an organic solvent. Thepreferred organic solvent is ethyl acetate although other organicsolvents can be used as would be obvious to the ordinarily skilledpractitioner in light of the disclosure herein. In other embodiments,the preferred organic solvent is isopropanol, a chloroform/Methanolmixture, or an equivalent thereof. The organic solvent is added to thelatex in a 1:1 proportion. In the preferred extraction process thesolvent latex combination is agitated.

The preferred agitation method is stirring although other agitationmethods are also contemplated to be effective. Following agitation, themixture is settled, or allowed to settle into distinct phases includingat least an organic layer and an aqueous layer. The organic phase orlayer is comprised largely of solute lipophilic materials, representingthe active constituent, and a significantly reduced quantity ofproanthocyanidin components relative to the pre-agitation step. Theorganic layer is separated from the aqueous layer for further processingpursuant to the preferred extraction process.

Moreover, it is common to find a gel-like substance in the organic layerat the interface of the aqueous and organic layers. This gel substanceis characterized as having a dark brown and purple color and compriseshydrophilic constituents trapped with water. In the preferred processthe gel substance is processed further to separate any active lipophilicconstituents from the hydrophilic constituents. The preferred manner ofprocessing the gel substance is the addition of a drying agent to theorganic layer or the gel substance. The preferred drying agent ismagnesium sulfate in a concentration of 0.5–5 g/L of contaminant gel. Itis contemplated that other equivalent drying agents at relativeeffective concentrations would also be effective and would be obvious tothe ordinarily skilled practitioner in light of the disclosure hereinand with undue experimentation.

The addition of the drying agent results in a precipitant, which trapswater and hydrophilic constituents or water-based colored chemicalcontaminants. The precipitant can be readily separated from thehydrophilic constituents by filtration or other techniques known toseparate precipitants. Actual laboratory procedures achieved acceptableresults using a Whatman #4 filter paper or an equivalent.

The steps of organic extraction, mixing with a drying agent andfiltration may be repeated up to three times to accomplish a thoroughextraction of the active lipophilic constituents. At this point in theprocess, the lipophilic materials are solutes contained within theorganic solvent, which are concentrated by evaporation of the solvent byone of several procedures, such as vacuum drying, freeze drying orheating. Actual heating up to 60 degrees Celsius produced acceptabledrying results.

The organic layer composition thus processed is rich in lipophilicmaterials but largely clear of hydrophilic contaminants. Following theextraction process, the color of the organic layer can be characterizedas a rose. Moreover, the reduced proanthocyanidin content isquantifiable spectrophotometrically. Relative absorbance of theextraction in the visible spectrum was compared to the absorbency peakof the parent latex (414 nm) in the visible range.

A product (CGO 110) of the extraction process disclosed herein is fromthe species Croton lechieri from the family Euphorbaceae. At aconcentration of 1 mg of extracted latex to 1 mL of water the disclosedprocess yielding the extraction (CGO 110) results in a 4.3 foldreduction in absorbance at 414 nm, as indicated in FIG. 1. Thisassessment was repeated 9 times with similar results achieved(significance difference P<0.0001, as denoted by the “*”). Similarlywhen sangre de grado or the extraction (CGO 110) at a concentrations of200 μg per mL of aloe vera gel were applied to aloe vera gel to mimictheir administration as topical products, there was also a significantlylower color response with the extracted sangre de grado, CGO 110 vs. theparent botanical (*P<0.0001). See FIG. 1. Estimates from the absorbencymeasurements indicate that the proanthocyanidin content was reduced byat least 90% relative to the nonextracted parent latex.

Extraction Process 2.

The latex from the Croton species is dried to its residual solid matterby methods such as heating, air-drying, vacuum or freeze-drying. Thedried latex is rich in proanthocyanidin compounds and thereforecharacterized by a dark burgundy color. To the dried latex matter theorganic solvent, ethyl acetate or an equivalent, is added. The driedlatex and organic solvent mixture is agitated and the organic solvent isremoved for further processing according to the procedure described inExample 1. This process may be repeated up to three times to accomplisha thorough extraction all lipophilic materials in the organic layer andsolvent. If any water bearing contaminants are present, the addition ofdrying agent followed by filtration as noted above, will remove thesecontaminants. Removing the ethyl acetate through various methodsincluding heating, air-drying, vacuum or freeze-drying then isolates thesolutes contained within this organic extract.

The extraction thus processed according to the disclosed processes ischaracterized by a significant reduction of proanthocyanidin compounds.The reduction of the proanthocyanidin compounds leaves the extractionsignificantly diminished in color producing compounds and yet amenableto health care applications.

Reduced Proanthocyanidin Content and Color Reactions

FIG. 1 illustrates the extent of proanthocyanidin depletion accomplishedby the extraction processes described herein. Relative absorbency offamily Euphorbaciae latex Sangre de Grado (SdG) is compared against asimilar quantity of the latex that has been processed according to oneof the procedures disclosed herein (CGO 110). As shown in FIG. 1, theextraction processes significantly diminishes the proanthocyanidincompounds or content compared to the parent latex material and confirmedby a significant (500%) reduction in absorbency in the 390 to 430 nmrange. Since this wavelength range is within the human visible range,the extraction representing a significant reduction in visible color ofthis organic extract compared to the parent material.

The presence of the proanthocyanidins in the parent latex provides arich burgundy color to the ethnomedicine, however it also results in thegeneration of an intense “chocolate” color when combined with variousbase vehicles, including Aloe barbadensis (aloe vera) gel—and can thusact to stain various materials and textiles. In contrast, the mixture ofthe organic extract (CGO 110) with a similar base vehicle significantlyreduces this color reaction, which can be readily quantifiedspectrophotometrically. FIG. 1 illustrates this result and compares, asimilar quantity of aloe barbadensis gel, which has insignificantabsorbency in the 390 nm to 430 nm range, mixed with a quantity of theparent latex (SdG Gel), and mixed with a similar quantity of parentlatex extracted by a process disclosed herein (CGO 110 Gel).

Sangre de Grado has potential benefits as a topical applicant forvarious inflamed, itchy and irritated dermatological conditions.However, its inherent color due to a high proanthocyanidin content andthus the generation of an intense coloring when combined with basevehicles hinders its use for these applications. As the proanthocyanidincontent and thus coloring are significantly reduced by the disclosedprocesses, alone or in combination with other topical crèmes, gels orbase vehicles, the extraction (CGO 110) signifies a marked improvementin the natural product and its uses.

Effects of the Organic Extract on Sensory Afferents

A prototypical activator of sensory afferent nerves, the nerves thatmediate the sensations of pain, itch, cough and nausea is capsaicin, thepungent chemical found in chili peppers. Activation of these nerves byan activator such as capsaicin leads to a multitude of responsesincluding vasodilation (mediated by the release of neurotransmittersfrom these activated nerves that cause blood vessels to relax),inflammatory cell recruitment, edema, and the sensations of pain anditching. The Extraction (CGO 110) was tested to determine its ability tosuppress sensory afferent nerve activation by testing its ability toinhibit capsaicin-induced increases in gastric blood flow.

The experiment involved the topical application of capsaicin to themucosal surface of the stomach in anesthetized rats and mucosal bloodflow measured by a laser Doppler flow meter. As indicated in FIG. 2, themarked increase in mucosal blood flow induced by 300 μM capsaicin wasprevented by either the parent material, SdG, or its organic extract,CGO 110 deplete of proanthocyanidins at doses of 2 and 0.2 mg/ml,respectively. Thus, the organic extract described in this applicationretains the ability to effectively prevent the activation of sensoryafferent nerves.

Effects on Morphine-Induced Emesis and Itch

Sangre de Grado has also been used ethnomedically for the treatment of avariety of intestinal complications including diarrhea, ulcerations,cancer and emesis. Using a well-established ferret model ofpost-operative complications of nausea, emesis and itch induced bymorphine, the extraction (CGO 110) was administered intraperitoneally (3mg/kg) to ferrets 15 minutes prior to the administration ofmorphine-6-glucuronide (M6G), known to promote itching, retching andvomiting. The animals were monitored for sixty minutes. As shown in FIG.3, the subcutaneous injection of 0.05 mg/kg M6G caused a significantnumber of vomiting (2.2±0.4) and retching (10±1.2) incidences in thecontrol group. In those animals treated with extraction (CGO 110), thenumber of these episodes was virtually abolished (vomiting 0.6±0.3;retching 2.2±0.7, P<0.05). Itch as indicated by licking responses wasreduced from a control value of 16.9±2.3 episodes to 2.2±0.7 in CGO 110treated animals (P0.05). Given the utility of this model to predicttreatments for itch, nausea and vomiting, the extraction (CGO 110)contains active components and is effective in the treatment of emesisand itch.

Cytotoxicity: Cancer Cell Selectivity

While Sangre de grado has traditional uses in the treatment of cancer,its utility is limited because it is equally toxic to both normal andcancerous cells. A process that could retain the ability of Sangre degrado to kill cancer cells but prevented these toxic effects on normalcells would represent a significant improvement over the traditionalmedicine and a benefit to the treatment of disease in both humans andanimals.

To test the selective cytotoxic ability of extraction (CGO 110) invitro, cancerous cells from the gastrointestinal tract (AGS: stomach)and both normal macrophages and normal intestinal epithelial cells(IEC-18) were utilized. Cancerous GI cells were chosen based on Sangrede grado's traditional application for gastrointestinal complications.Cell death was determined by the MTT assay[3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide], whichassesses cell number by virtue of its oxidative or respiratory activityand the generation of a dye detectable at a wavelength of 550 nm.

As shown in FIG. 4, in normal cells, Sangre de grado caused significantcell death in both macrophages and IEC-18 cells while the sameconcentrations of the organic extract CGO 110 did not. From this we candetermine that the lipidic extract CGO 110 has improved safety over theparent botanical. Treatment of stomach cancer cells (AGS) with both CGO110 and Sangre de grado caused cytotoxicity (cell death), and thelipidic extract, CGO 110, was more potent than the parent botanical [the“*” in FIG. 4 denotes a significant difference between the Sangre degrado and organic extract CGO 110 formulations (P<0.05)]. Collectively,these results indicate that CGO 110 is selectively cytotoxic tocancerous cells compared to the parent botanical, thereby representing amarked improvement in safety.

Although the invention has been described in detail with reference toone or more particular preferred embodiments, persons possessingordinary skill in the art to which this invention pertains willappreciate that various modifications and enhancements may be madewithout departing from the spirit and scope of the claims that follow.

1. A method of extracting lipophilic components from Croton lechiericomprising; combining plant material from Croton lechieri with anorganic solvent; agitating the combination; settling the combinationinto distinct phases to resolve a layer substantially comprised ofhydrophilic constituents and an organic layer substantially comprised ofthe lipophilic constituents; separating the organic layer from the layersubstantially comprised of hydrophilic constituents; and evaporating theorganic solvent from the organic layer to resolve the lipophilicconstituents.
 2. The method of claim 1 wherein, the plant material islatex.
 3. The method of claim 1 wherein the organic solvent is selectedfrom the group consisting of ethyl acetate, isopropanol andchloroform/Methanol mixture.
 4. The method of claim 1 furthercomprising, adding a drying agent to the settled organic layer prior tothe step of evaporating the organic layer to further precipitate anyremaining hydrophilic constituents; and filtering the organic layer toresolve the lipophilic constituents.
 5. The method in claim 4 whereinthe drying agent selected from the group consisting of magnesium sulfateand sodium sulfate.
 6. The method in claim 5 wherein the drying agent ismagnesium sulfate and the amount added per liter of organic layer isbetween about five hundred milligrams (500 mg) to five grams (5 g) perliter.
 7. The method in claim 4 wherein, after the step of filtering theorganic layer, the organic layer at a concentration of one milligram permilliliter (1 mg/mL) of 50% (v/v) ethanol/water has an absorbance ofabout 0.120 Abs Units in the wavelength range between about 390 nm andabout 430 nm, relative to an absorbency of about 515 Abs Units withinthe same wavelength range.
 8. The method of claim 4 wherein thehydrophilic constituents having proanthocyanidin components are reducedby at least about 90% relative to the parent latex.
 9. The method ofclaim 1 wherein the step of evaporating the precipitate to resolve thehydrophilic constituents is selected from the group of evaporatingmethods consisting of evaporation, spray drying, freeze drying, andvacuum drying.
 10. A method for making an extract from Croton lechieri;combining plant material from Croton lechieri with an organic solvent;agitating the combination; settling the combination into distinct phasesto resolve a layer predominantly comprised of hydrophilic constituentsand an organic layer substantially comprised of the lipophilicconstituents; separating the organic layer from the layer substantiallycomprised of hydrophilic constituents; and evaporating the organicsolvent from the organic layer to resolve the lipophilic constituents.11. The method of claim 10 wherein, the organic solvent is selected fromthe group consisting of ethyl acetate, isopropanol andchloroform/Methanol mixture.
 12. The method of claim 10 furthercomprising, adding a drying agent to the settled organic layer prior tothe step of evaporating the organic layer to further precipitate anyremaining hydrophilic constituents; and filtering the organic layer toretain the lipophilic constituents.
 13. The method in claim 12 whereinthe drying agent selected from the group consisting of magnesium sulfateand sodium sulfate.
 14. The method in claim 13 wherein, the drying agentis magnesium sulfate and the amount added per liter of organic layer isbetween about five hundred milligrams (500 mg) to five grams (5 g) perliter.
 15. The method in claim 12 wherein, after the step of filteringthe organic layer, the organic layer at a concentration of one milligramper milliliter (1 mg/mL) of 50% (v/v) ethanol/water has an absorbance ofabout 0.120 Abs Units in the wavelength range between about 390 nm andabout 430 nm, relative to an absorbency of about 515 Abs Units withinsaid wavelength range.
 16. The method of claim 12 wherein, thehydrophilic constituents comprising proanthocyanidin components arereduced by at least about 90% relative to the parent latex.
 17. Themethod of claim 10 wherein, the step of evaporating the precipitate toresolve the hydrophilic constituents is selected from the group ofevaporating methods consisting of evaporation, spray drying, freezedrying, and vacuum drying.
 18. An extract from Croton lechleri made bythe process of claim 12 wherein, UV absorbency of the extract between arange of 390 nm and 430 nm is reduced by at least one-half relative tothe absorbency for unextracted plant material within said range.
 19. Anextract as in claim 18 wherein, the UV absorbency of the extract betweena range of 390 nm and 430 nm is about 0.010 Abs Units relative to about0.030 Abs Units for unextracted plant material within said range.